The Myoglobin ELISA kit uses a unique monoclonal antibody directed against a distinctantigenic determinant on the myoglobin molecule. This antibody is used for solid phase immobilization (on microtiter wells). A goat anti-myoglobin Ab is conjugated to HRP and used for detection. The test sample is allowed to react simultaneously with the two antibodies, resulting in the myoglobin molecules being sandwiched between solid phase and HRP-linked antibodies. After a 45 minute incubation period at room temperature, the wells are washed with water. A TMB reagent is then added and the reaction stopped after 20 minutes by addition of 1N HCl. The absorbance value at 450 nM (or 405 nM) is determined using a 96-well microtiter plate reader. Human Myoglobin concentration is directly proportional to A450 (or A405) and is determined by reference to human myoglobin standards (0, 25, 100, 250, 500, and 1000 ng/mL) supplied in liquid, pre-diluted, ready to use form with the kit.

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